Severe to profound congenital hearing loss affects approximately 1/1000 newborns in developed countries (Morton, in The Genetics of Hearing Impairment, The New York Acad. Sci., New York, 630, pp. 16-31 (1991) and Gorlin et al., Hereditary hearing loss and its syndromes, New York: Oxford University Press (1995)). At least half of all congenital hearing loss is due to inherited disorders, while the remaining cases are likely due to environmental factors such as acoustic trauma, ototoxic drug exposure, or bacterial or viral infection (ACMG, Genetics in Medicine, 4: 162-171 (2002)). Approximately 70% of inherited, congenital hearing loss is nonsyndromic (no additional symptoms present) (Steel and Kros, Nat. Genet. 27: 143-149 (2001)). Typically, nonsyndromic hearing impairment (NSHI) is attributable to sensorineural anomalies (ACMG, supra.).
Consistent with the complexity of the cochlear apparatus itself, which consists of over 1 million moving parts (Hudspeth, Science 230: 745-752 (1985)), over 77 loci associated with NSHI have been mapped to human chromosomes, and more than 50 of these have been sequenced (ACMG, supra.). These genes are subdivided based on their mode of inheritance. Deafness genes are named DFNA if autosomal dominant (22% of characterized loci), DFNB, if autosomal recessive (77% of characterized loci), or DFN if X-linked (1% of characterized loci) (ACMG, supra.). The majority of sensorineural NSHI appears to be monogenic (ACMG, supra.). Despite the diversity of associated loci, more than 50% of cases are due to autosomal recessive variations in a single gene, DFNB1, also known as connexin 26 (Cx26) and gap-junction beta 2 (GJB2) (reviewed in Kenneson, A. et al., Genetics in Medicine 4: 258-274 (2002)).
The connexin 26 protein was identified in 1989 in liver tissue (Zhang and Nicholson, J Cell Biol. 109: 3391-401 (1989)) and later associated with NSHI as DFNB1 (Guilford, et al., Nature Genet. 6: 24-8 (1994)). The gene is located on chromosome 13q11-12 (Mignon, C. et al., Cytogenet Cell Genet 72: 185-6 (1996)). Connexin 26 is a member of the connexin family of gap junction proteins, which are cellular channels that enable intercellular communication by spanning plasma membranes and linking the cytoplasm of adjacent cells (White, Brain Research Reviews 32: 181-183 (2000)). Each such channel is composed of two connexons, each of which in turn is an assembly of six connexin subunits. The connexin 26 protein is expressed in two groups of cochlear cells in the inner ear: non-sensory epithelial cells and connective tissue cells (Kelley et al., Am. J. Hum. Genet. 62: 792-799 (1998)). The majority of deafness-causing mutations of the connexin 26 gene are autosomal recessive, loss-of-function alleles that result in prematurely truncated protein. The loss of functional connexin 26 protein likely disrupts potassium ion flow within the cochlea, thus interfering with the normal functioning of hair cells and sensorineural function (reviewed in Lefebvre, Brain Research Reviews, 32: 159-162 (2000)).
Despite the identification of more than 90 variants in the connexin 26 protein, most of which result in amino acid changes, the majority of NSHI cases attributable to mutations in this gene result from just a handful of alleles, which in turn are population-based (Kenneson, A. supra.). A single base deletion of a G residue, alternately referred to as 30ΔG or 35ΔG, has a prevalence of 1-3% in Caucasian populations and accounts for ⅔ of cases of congenital deafness in these populations. By itself, this allele accounts for as much as 10% of all childhood hearing loss (Kelley et al., Brain Research Reviews, 32: 184-188 (2000) and Kelley, P. M. et al. Am. J. Hum. Genet. 62: 792-99 (1998)). This mutation results in the loss of one of six contiguous G residues in the upstream portion of the gene and results in prematurely truncated protein (White, Brain Research Reviews 32: 181-183 (2000)). This sequence appears to be a mutational hot spot and is similar to a sequence predicted to be a site of frequent DNA replication errors (Kelley, supra). Similarly, as many as 10% of Ashkenazi Jews carry a deletion of a T residue at 167 (167ΔT), while 1% of the Japanese and Korean populations carry 235ΔG. Preliminary studies suggest that penetrance of hearing loss in individuals homozygous for these alleles is complete in these populations (Kenneson, supra.).
A combination of factors, i.e. increased frequency of newborn hearing screening, improved accuracy of testing (Norton et al., Ear Hear, 21: 529-535 (2000)), and the demonstration that early detection of hearing loss coupled with intervention results in the development of language skills among pre-lingual deaf children that approach those of hearing children (Yoshinaga-Itano, et al., Pediatrics, 102: 1161-1171(1998) and Moeller, Pediatrics, 106: 3 e43 (2000)), has led to an increase in the use of genetic testing in follow-up analysis of infants with hearing loss as well as in the use of genetic counseling for families with hearing loss-affected offspring (ACMG, supra.). Molecular genetic methods applied to the analysis of connexin 26, or GJB2 mutations include the following (reviewed in Kenneson, supra): sequencing; allele-specific PCR, often followed by sequencing of positives; PCR-RFLP; PCR-SSCP of exons 1 and 2; PCR-DGGE; PCR with allele-specific probe hybridization; heteroduplex analysis followed by sequencing of positives. The majority of these methods, including methods described in U.S. Pat. Nos. 5,998,147 and 6,485,908 rely on PCR, and many require gel electrophoresis to discriminate the presence of variants. Moreover, with the exception of those approaches that involve sequencing, these methods do not distinguish between true heterozygotes and compound heterozygotes, i.e. individuals carrying two different variant alleles.
Given the potential importance of analysis of the connexin 26 and GJB2 genes in broad-based genetic testing for NSHI, there is a need for detection assays that may be applied directly to the analysis of connexin 26 and GJB2 sequences (e.g. genomic sequences), as well as assays that can be used to analyze large numbers of samples, i.e. high-throughput assays.